Design

A comparative in vitro cell culture experiment.

Time and setting

The experiment was performed at the Laboratory of Liaoning Medical College, China, from December 2009 to July 2011.

Materials 

Thirty-five, neonatal (≤ 24 hours) Sprague-Dawley rats of  specific pathogen free grade, weighing 15 ± 5 g, were purchased from the Medical Laboratory Animal Center of Liaoning Medical College, China, with permission No. SCXK (Liao) 2003-0007. All experimental protocols were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[33].

Methods

Isolation and primary culture of rat cortical neurons

Neonatal rats were soaked in 75% (v/v) ethanol for     1 minute and subsequently decapitated. The heads were pre-cooled in D-Hank’s buffer, and the skull and meninges were removed under a microscope (Olympus, Tokyo, Japan), followed by separation of the cortex. Cortical tissue was washed three times in ice-cold D-Hank’s buffer, and micro-scissors were used to carefully remove the tissue, which was placed in 2.5 g/L trypsin (Sigma, St. Louis, MO, USA) to digest for      10 minutes at 37°C. Digestion was terminated with fetal calf serum, followed by three washes in ice-cold D-Hank’s buffer. Culture plate processing: Glass coverslips were placed into 6-well culture plates 1 day prior to cell culture. A total of 1 mL polylysine (0.01% (w/v)) was added to each well and allowed to incubate overnight, followed by draining and drying^[34]. The wells were rinsed twice with D-Hank’s buffer, followed by an appropriate amount of culture medium, and were allowed to reach the proper temperature (37°C) inside the incubator^[35]. Serum-free medium, Neurobasal-A (Gibco; consisting of 2% (v/v) B27, 10 mM hydroxyethyl piperazine ethanesulfonic acid, 2 mM L-glutamine,    25 μM glutamate, and 1% (v/v) penicillin-streptomycin), was added and the cell suspension was gently triturated and then pushed through a mesh filter. A sample of the cell suspension (50 μL) was combined with 150 µL 2% (v/v) trypan blue (Wuhan Boster, Wuhan, China) and  200 µL PBS, mixed, and counted using a cell counter (Beijing Biosynthesis Biotechnology, Beijing, China). The number of living and dead cells was quantified under the microscope, revealing that > 70% were living cells^[36]. Cells (1 × 10⁶/mL) were incubated in 2-mL culture medium at 37°C and maintained in 5% CO₂ for 24 hours, after which the medium was replaced and non-adherent cells were discarded. The medium was replaced every  3 days. Cells were continuously cultured for 7 days and used for further experiments.

Identification of cortical neurons by MAP-2 and NSE

MAP-2 and NSE primary and secondary antibodies, purchased from Sigma, were utilized for immunocytochemistry staining. Positive cells exhibited green and brown staining under the light microscope^[37]. Primary cultured cortical neurons, which were in an active growth phase, were digested and seeded onto cover slips in 6-well culture plates. The medium was removed when the cells reached 70–80% confluency. The cells were then placed in induction medium containing β-mercaptoethanol (Sigma) (induction for 1, 3, 5, and 8 hours). The coverslips were then removed from the culture plates and washed three times with PBS. The cells were fixed with 4% (w/v) paraformaldehyde for   30 minutes and washed three times with PBS (5 minutes each). After discarding the fixative solution, cells were incubated in 0.1% (v/v) Triton-X 100 for 8 minutes, washed three times with PBS for 3 minutes each, incubated in 3% (v/v) H₂O₂ for 10 minutes to eliminate endogenous peroxidase, and washed three times with PBS for 3 minutes each. The cells were then incubated in working solutions of primary monoclonal antibodies: rabbit anti-MAP-2 (1:100) and rabbit anti-NSE (1:100) at 4°C overnight. The cells were washed three times with PBS for 3 minutes each, followed by incubation in secondary antibodies: goat anti-mouse IgG-fluorescein isothiocyanate (1:100; MAP-2 staining) and mouse anti-rabbit IgG (1:100; NSE) at room temperature for   15 minutes, and washed three times with PBS for 3 minutes each. Images were observed and collected under a fluorescence microscope (MAP-2, × 400; Leica, Solms, Germany) using an excitation wavelength of 495 nm and an absorption wavelength of 520 nm, and under a microscope (NSE, × 400; Nikon, Tokyo, Japan).

Experimental grouping and treatments

Cells were randomly divided into six groups: sham group, NMDA group, 0.5 μM KN-93 group, and an NMDA in combination with three different concentrations of KN-93 (0.25, 0.5, 1.0 μM; Sigma) groups. Cells (1 × 10⁶/mL) were seeded and cultured for 7 days and then were treated with NMDA (50 μM) alone and in combination with KN-93 (0.25, 0.5, 1.0 μM) after NMDA treatment for 30 minutes, excepting the sham group, which was treated with an equal volume of PBS, and the 0.5 μM KN-93 group, which was treated with 0.5 μM KN-93 only. Twenty-four hours later, identification of cortical neurons, detection of neuronal viability by MTT, TUNEL and PI double staining, and western blot analysis were carried out.

Neuron viability by MTT assay

Cell viability of cortical neurons was assessed by the MTT assay^[38-39]. After exposure to NMDA or NMDA combined with KN-93 for 24 hours, DMEM and freshly dissolved MTT (aseptic, 5 mg/mL; Beijing Biosynthesis Biotechnology) was added to culture plates at a final concentration of 10% (w/v). Cells were then returned to the incubator for 4 hours. Dimethyl sulfoxide (200 μL) was added to the plates and agitated for 10 minutes at 37°C to solubilize the MTT formazan crystals. The absorbance value at 570 nm was read spectrophotometrically (Bio-TEK Instruments, Inc., Winooski, VT, USA).

Evaluation of LDH leakage

Cell membrane permeability was determined by measuring LDH release from neurons into the incubation medium using a colorimetric assay kit (Wuhan Boster, Wuhan, China) where the absorbance of the produced formazan (measured at 500 nm) product is proportional to LDH activity. Total LDH was determined after adding a final concentration of 10% (v/v) Triton X-100 and disrupting the slices by homogenization with a Tissue Tearor. Differences related to neuron size were minimal; therefore, data from different experiments were pooled by normalizing the amount of LDH activity released from disrupted neurons.

Apoptosis measured by TUNEL and PI double staining

Neuronal apoptosis in cultured cortical neurons was examined by TUNEL using a commercially available kit (Beijing Biosynthesis Biotechnology), which relies on enzymatic labeling of DNA strand breaks. Cortical neurons were plated onto 15-mm diameter, 1-mm thick glass cover slips precoated with poly-L-lysine. The cells were fixed with 4% (w/v) paraformaldehyde in PBS at 20°C for 1 hour on a slide. The slide was incubated with blocking solution for 10 minutes at 20°C to eliminate endogenous peroxidase, followed by incubation in 0.1% (v/v) Triton X-100 in PBS for 2 minutes on ice. The sample was added to the TUNEL reaction mixture, which was incubated in a humidified atmosphere in the dark for    1 hour at 37°C. Converter-peroxidase (50 µL; Roche Applied Science Company, Basel, Switzerland) was added to the sample, and after incubating the reaction at 37°C for 30 minutes, the sample was treated with 1 mL substrate solution for 15 minutes. TUNEL-positive cells were counted using a cell counter (Shanghai Aibo Medical Device Company, Shanghai, China). For PI staining, cells were cultured in plates for 24 hours, and then treated with PI (Sigma; 50 g/mL) dissolved in PBS (pH 7.4) containing 0.1%Triton X-100 (v/v) and 100 g/mL RNase. Apoptotic cell suspensions were prepared at a concentration of 10⁶/mL. The cell suspension (1 mL) was mixed with 10 µL PI for 10 minutes and 5 mL PBS was added. The suspension was centrifuged at 340 × g for  5 minutes, the supernatant was discarded, and cells were washed twice. Viable neurons were quantified by counting the number of PI-positive cells under a fluorescence microscope (Leica, Solms, Germany) at 536 nm. The results were expressed as a ratio of positive cells of TUNEL to PI^[40-41].

Detection of intracellular calcium concentration in cultured cortical neurons

Cell suspensions of cortical neurons were pre-warmed at 37°C for 5 minutes. Fura-2/AM (Beijing Biosynthesis Biotechnology) was added into the suspension and the mixture was left at 37°C for 30 minutes. Cells were then centrifuged at 150 × g for 5 minutes. After the supernatant was discarded, the suspension was washed twice in D-Hanks solution and the cell concentration was adjusted to 1 × 10⁶/mL. 1 µL of cell suspension was put into the colorimetric plate (Yixing Ye Hui Glass Instrument Factory, Yixing, Jiangsu Province, China) cuvette and the concentration of intracellular calcium was detected with a fluorescence spectrophotometer (Tianjin Port East Science Technology Development Company Ltd., Tianjin, China). Upon binding Ca²⁺, the excitation spectrum of Fura-2 shifted to shorter wavelengths between 300 nm and 400 nm, while the peak emission remained steady at around 510 nm. Peak fluorescence intensity occurred at 345 nm, which meant that Fura-2/AM had entered cortical neurons. The concentration of cytoplasmic calcium was continuously monitored at 345 nm. The concentration of intracellular calcium was calculated by measuring the fluorescence (F) of solutions with different Ca²⁺ concentrations using the equation: [Ca²⁺] = K_d(F₀ – F_min)/(F_max – F₀), where Kd was the dissociation constant of the chemical reaction for Ca²⁺ buffering by the fluorescent dye and F₀, F_max and F_min represented the instantaneous, maximal and minimal dye fluorescence emissions, respectively^[42]. The value of K_d was 224 nm. These values were determined with internal solutions containing 10 mM ethylene glycol tetraacetic acid (0 Ca²⁺), and 10 mM CaCl₂ (max Ca²⁺). 

Expression of procaspase-3, caspase-3, P-CaMKII and total-CaMKII levels

Procaspase-3 and caspase-3 detection: Cells were homogenized in lysis buffer containing 250 mM sucrose and 15 nM digitonin, and centrifuged at 150 × g for     1 minute. The pellet obtained was solubilized with sodium dodecyl sulfate-stopping solution (4% (w/v) sodium dodecyl sulfate, 2 mM ethylenediaminetetraacetic acid, 8% (v/v) β-mercaptoethanol, and 50 mM Tris, pH 6.8). The supernatant achieved from the digitonin lysis buffer, which was considered the cytosolic fraction, was lyophilized and then resolubilized in sodium dodecyl sulfate-stopping solution. Samples (50 µg of total protein/track) were separated by SDS-PAGE using a 14% (w/v) gel. For procaspase-3 and caspase-3 analysis, cells were solubilized with sodium dodecyl sulfate- stopping solution and samples (30 µg of total protein/track) were separated by SDS-PAGE using a 12% (w/v) gel. The amount of protein loading was controlled by Coomassie staining of gels or Ponceau staining of the nitrocellulose membranes^[43]. Protein was transferred to nitrocellulose membranes using a    400 mA current (3 hours, 4°C). The membranes were blocked with 5% (w/v) non-fat dry milk, followed by a second block of 2.5% (w/v) gelatine both in Tris- buffered saline (10 mM Tris, 150 mM NaCl, pH 7.5). All steps were followed by three washes with Tris-buffered saline-Tween 20 (0.05% (v/v) Tween-20, 10 mM Tris, 150 mM NaCl, pH 7.5). Anti-procaspase-3 and caspase-3 antibodies (1:500; oncogene, Cambridge, MA, USA) were used to detect specific proteins.

P-CaMKII and total-CaMKII detection: Samples containing membrane protein were denatured for    10 minutes at 95–98°C and electro-transferred to nitrocellulose membrane with 10% (w/v) Tris solution. The membranes were blocked in the buffer (5% (w/v) non-fat dry milk in PBS) at room temperature for      2 hours, and then incubated with the monoclonal antibody anti-rat P-CaMKII (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-total- CaMKII (1:500, Santa Cruz Biotechnology) at 4°C overnight. The membrane was then washed for      10 minutes each, three times using blocking buffer and incubated with 1:500 diluted anti-mouse IgG labeled with the horseradish peroxidase conjugate (Beijing Zhongshan Biotechnology Co., Ltd, China) for 1 hour at room temperature. Washing was repeated and protein signals were visualized using an enhanced chemiluminescence system (Amersham, Cambridge, UK) on an X-ray film and analyzed on the Gel Image Analysis System (Bio-Rad, Hercules, CA, USA). The levels of procaspase-3, caspase-3, P-CaMKII and total-CaMKII expression were determined by calculating the ratio of their absorbance values relative to β-actin.

Statistical analysis

Data were expressed as mean ± SD and analyzed using SPSS software (Windows version 15.0; SPSS, Chicago, IL, USA). Statistical analysis for comparisons among six groups was performed using one-way analysis of variance. A P value less than 0.05 was considered statistically significant.

KN-93, a calmodulin-dependent protein kinase II inhibitor, has a neuroprotective effect, and its underlying mechanism of action may be related to the down-regulation of caspase-3 and calmodulin-dependent protein kinase II expression.
1.钙调蛋白激酶II在认知功能损害中的作用已有很多报道，如血管性痴呆、颞叶癫痫所致的认知功能障碍，与凋亡通路的具体联系仍处于实验阶段。
2.实验应用N-甲基-D-天冬氨酸对原代培养的SD乳鼠大脑皮质神经元造成损伤，然后应用钙调蛋白激酶II抑制剂KN-93进行干预。
3.实验发现，抑制损伤神经元钙调蛋白激酶II表达的同时，细胞凋亡也减少。